Introduction
1
Pulsed Multiple Reaction Monitoring Mode: The Novel Sensitive Approach for Biomolecule Quantitation Mikhail Belov, Satendra Prasad, David Prior, William Danielson, Karl Weitz, Vladislav Petyuk, Yehia Ibrahim and Richard Smith Pacific Northwest National Laboratory, Richland, WA Introduction Overview Methods Results Acknowledgements Portions of this work were supported by the National Center for Research Resources, grant RR 18522 and National Cancer Institute, grant R33 CA12619-01. Samples were analyzed using capabilities developed under the support of the NIH National Center for Research Resources (RR18522) and the U.S. Department of Energy Biological and Environmental Research (DOE/BER). Significant portions of the work were performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated for the DOE by Battelle under contract DE-AC05- 76RLO-1830. References 1. Belov, M.E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev, A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 19-23 2. Wouters, E. R.; Splendore, M.; Mullen, C.; Schwartz, J. C.; Senko, M. W.; Dunyach, J. J. 57 th Conference of American Society for Mass Spectrometry; 2009. 3. Ibrahim, Y.; Belov, M. E.; Tolmachev, A. V.; Prior, D. C.; Smith, R. D. Anal. Chem., 2007, 79, 7845-7852. 4. Clowers, B.H.; Ibrahim, Y.M.; Prior, D.C., Danielson, W.F., Belov, M.E.; Smith R.D. Anal. Chem., 2008, 80, 612- 623. Conclusions CONTACT: Mikhail Belov, Ph.D. Biological Sciences Division, K8-98 Pacific Northwest National Laboratory P.O. Box 999, Richland, WA • This work reports on the improved limit of detection of a liquid chromatography (LC)-triple quadrupole instrument operating in the multiple reaction monitoring mode (MRM) by incorporation of an ion funnel trap (IFT) between an ion source and a quadrupole analyzer. • Incorporation of an ion funnel trap ( IFT) into a triple quadrupole analyzer operating in the multiple reaction monitoring mode (MRM) resulted in 3 to 10 fold improvement in the limit of detection (LOD) as compared to the ion funnel interface and 20 to 50 fold LOD improvement in comparison to that of the commercial instrument. • Rigorous studies of signal intensities of peptides added to a highly complex biological matrix at concentrations ranging from 0.5 nM to 1000 nM showed the linear response of the LC-IFT-MRM instrument with respect to the concentrations of low abundance peptides. Multiple Reaction Monitoring (MRM) offers a highly sensitive analytical platform to quantify trace constituents in complex biological matrices by selectively delivering analyte ions from an ESI source to an MS detector. Further sensitivity improvements with LC-MRM approach are achievable by enabling higher efficiency transport of analyte components from an ion source to the MS analyzer, eliminating dead times in analyses of fragment ions and reducing background ion signals. Electrodynamic ion funnel (IF) 1 or S- lense 2 have been shown to drastically improve MS sensitivity. A recently introduced ion funnel trap (IFT) has been demonstrated to further enhance the limit of detection for both the time-of-flight 3 and ion mobility time- of-flight mass spectrometers 4 . The premise for sensitivity improvement with the IFT coupled to a triple quadrupole instrument is due to: i) ion accumulation in the RF- energized trap, which facilitates improved droplet desolvation manifested in the reduced background ion noise at the detector, ii) enhancement in signal amplitude for a given transition because of an order- of-magnitude increase in the ion charge density per unit time compared to the continuous mode of operation, and iii) the unity duty cycle in Chemicals and Materials: Lyophilized Kemptide, Angiotensin I, Syntide 2, Bradykinin, Leucine and Enkephalin, Dynorphin A Porcine 1-13, Neurotensin, and Fibrinopeptide A were purchased from Sigma-Aldrich (St. Louis, MO). These were serially diluted to prepare concentrations ranging from 0.25 nM to 500 nM in 0.25 mg/mL of tryptic digest of Shewanella oneidensis MR-1 proteins. Figure 1. Experimental setup and graphical interface of instrument control software for an ion funnel trap Figure 2. Pulsing sequence of experiment with the ion funnel trap. Ions are accumulated in the trap during dead time and dwell time followed by a 0.5 ms release event. Ion release event is synchronized with the start of Q3 scan. Parent Ions Ion Trans. Frag. Trans . Frag. Trans . Frag. CE Kemptide 386.74 (2+) 409.27 b 3 -NH 3 539.34 a 5 -NH 3 567.33 b 5 -NH 3 25 Angiotensin I 432.90 (3+) 534.27 b 4 619.36 a 5 647.35 b 5 21 Syntide 2 503.32 (3+) 283.18 b 3 429.28 y 4 705.94 y 14 2+ 23 Bradykinin 530.79 (2+) 522.27 y 9 2+ -NH 3 710.36 y 6 807.42 y 7 30 Dynorphin A Porcine 1-13 535.34 (3+) 455.21 y 11 3+ - NH 3 529.70 y 13 3+ - NH 3 712.68 y 12 3+ - NH3 28 Leucine Enkephalin 556.28 (+) 278.11 b 3 397.19 a 4 425.18 b 4 22 Neurotensin 558.31 (3+) 578.85 y 9 2+ 643.73 y 10 2+ 725.90 y 11 2+ 26 Fibrinopeptide A 768.85 (2+) 445.25 y 5 645.33 y 7 1077.5 3 y 11 29 Electroni cs to control IF and IFT Source interfa ce TSQ 4 column HPLC Timing pulse to synchronize trap release and Q3 scan Voltages to control trap Transiti on 1 Transiti on 2 Dead time Dead time Dead time accumulat ion accumulat ion accumulat ion ejec t ejec t ejec t Entrance grid Exit grid Transitio n 3 Q3 Trigger Exit grid Entrance grid ION FUNNEL TRAP Q1 Q3 Collision cell Q2 Detector ION FUNNEL 10 torr 1 torr 1.5x10 -3 torr multi inlet -1 -0.6 -0.2 0.2 0.6 1 0 5 10 15 20 25 RETENTIO N TIM E,m in IN TEN SITY IO N FUNNEL TRAP 2 nM Anigiotensin I in 0.25 m g/m L Shew anella digest IO N FUNNEL 2 nM Anigiotensin I in 0.25 m g/m L Shew anella digest 0 5 10 15 20 25 30 35 0.000E+00 1.000E+08 2.000E+08 3.000E+08 4.000E+08 5.000E+08 6.000E+08 R² = 0.994070172854722 Kemptide fmoles loaded onto LC column Peak Area 0 5 10 15 20 25 30 35 0.00E+00 1.00E+08 2.00E+08 3.00E+08 4.00E+08 5.00E+08 6.00E+08 R² = 0.997333156050591 Angiotensin I fmoles loaded onto LC column Peak Area 0 5 10 15 20 25 30 35 0.00E+00 5.00E+08 1.00E+09 1.50E+09 2.00E+09 2.50E+09 R² = 0.996003925872965 Leucine Enkephalin fmoles loaded onto LC column Peak Area Peak Area 0 5 10 15 20 25 30 35 0.00E+00 1.00E+08 2.00E+08 3.00E+08 4.00E+08 5.00E+08 6.00E+08 7.00E+08 R² = 0.994467325150107 fmoles loaded onto LC column Fibrinopeptide A 5 10 15 20 25 30 35 0E+00 1E+08 2E+08 3E+08 4E+08 5E+08 6E+08 R² = 0.998842838158562 Neurotens in fmoles loaded onto LC column Peak Area 0 5 10 15 20 25 30 35 0.00E+00 4.00E+07 8.00E+07 1.20E+08 1.60E+08 2.00E+08 R² = 0.99828682610235 fmoles loaded onto LC column Bradykini n Peak Area Figure 3. LC-MS/MS experiment with a 0.25 mg/mL Shewanalla oneidensis digest spiked with nine peptides (see table 1). Inset shows three transitions of neurotensin acquired in continuous (blue trace) and trapping (red trace) modes SNR SNR kemptide Leucine Enkephalin moles moles Figure 4. Signal-to-Noise (SNR) of kemptide and leucine enkephalin as a function of the peptide amount added to a 0.25 mg/mL Shewanella oneidensis digest in LC-MRM experiments. Bottom panels show selected ion chromatograms representing three transitions of the two peptides and the matrix signal in the corresponding m/z ranges. Table 1. List of peptides and the corresponding fragments monitored in LC-MRM experiments with a 0.25 mg/mL Shewanella oneidensis digest 0 20000 40000 60000 80000 534.265 534.266 534.267 534.268 m/z IN TEN SITY Angiotensin I,b 4 +,m /z 534.27 IO N FUNNEL TRAP IO N FUNNEL 0 20000 40000 60000 80000 619.356 619.357 619.358 619.359 m/z IN TEN SITY Angiotensin I,a 5 +,m /z 619.36 IO N FUNNEL TRAP IO N FUNNEL 0 20000 40000 647.351 647.352 647.353 647.354 m/z IN TEN SITY A ngiotensin I,b 5 +,m /z 647.36 IO N FUNNEL TRAP IO N FUNNEL Figure 5. Comparison of the selected ion chromatograms corresponding to the three transitions of Angiotensin I added with other 8 peptides to a 0.25 mg/mL digest of Shewanella oneidensis in LC-MRM experiments. Trapping time in the IFT was 40 ms. Figure 6. Peak area as a function of the peptide amount for several peptides added to a 0.25 mg/mL Shewanella oneidensis digest in LC-IFT-MRM experiments.
description
Dead time. Dead time. Dead time. Transition 3. Transition 1. Transition 2. Q3 Trigger. eject. eject. eject. Leucine Enkephalin. kemptide. Entrance grid. SNR. SNR. Exit grid. accumulation. accumulation. accumulation. Collision cell Q2. ION FUNNEL TRAP. ION FUNNEL. Q1. Q3. - PowerPoint PPT Presentation
Transcript of Introduction
Pulsed Multiple Reaction Monitoring Mode: The Novel Sensitive Approach for Biomolecule Quantitation
Mikhail Belov, Satendra Prasad, David Prior, William Danielson, Karl Weitz, Vladislav Petyuk, Yehia Ibrahim and Richard SmithPacific Northwest National Laboratory, Richland, WA
Introduction
OverviewMethods Results
AcknowledgementsPortions of this work were supported by the National Center for Research Resources, grant RR 18522 and National Cancer Institute, grant R33 CA12619-01. Samples were analyzed using capabilities developed under the support of the NIH National Center for Research Resources (RR18522) and the U.S. Department of Energy Biological and Environmental Research (DOE/BER).
Significant portions of the work were performed in the Environmental Molecular Science Laboratory, a DOE/BER national scientific user facility at Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is operated for the DOE by Battelle under contract DE-AC05-76RLO-1830.
References1. Belov, M.E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Tolmachev,
A. V.; Prior, D. C.; Harkewicz, R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 2000, 11, 19-23
2. Wouters, E. R.; Splendore, M.; Mullen, C.; Schwartz, J. C.; Senko, M. W.; Dunyach, J. J. 57th Conference of American Society for Mass Spectrometry; 2009.
3. Ibrahim, Y.; Belov, M. E.; Tolmachev, A. V.; Prior, D. C.; Smith, R. D. Anal. Chem., 2007, 79, 7845-7852.
4. Clowers, B.H.; Ibrahim, Y.M.; Prior, D.C., Danielson, W.F., Belov, M.E.; Smith R.D. Anal. Chem., 2008, 80, 612-623.
Conclusions
CONTACT: Mikhail Belov, Ph.D.Biological Sciences Division, K8-98Pacific Northwest National LaboratoryP.O. Box 999, Richland, WA 99352E-mail: [email protected]
• This work reports on the improved limit of detection of a liquid chromatography (LC)-triple quadrupole instrument operating in the multiple reaction monitoring mode (MRM) by incorporation of an ion funnel trap (IFT) between an ion source and a quadrupole analyzer.
• Incorporation of an ion funnel trap ( IFT) into a triple quadrupole analyzer operating in the multiple reaction monitoring mode (MRM) resulted in 3 to 10 fold improvement in the limit of detection (LOD) as compared to the ion funnel interface and 20 to 50 fold LOD improvement in comparison to that of the commercial instrument.
• Rigorous studies of signal intensities of peptides added to a highly complex biological matrix at concentrations ranging from 0.5 nM to 1000 nM showed the linear response of the LC-IFT-MRM instrument with respect to the concentrations of low abundance peptides.
Multiple Reaction Monitoring (MRM) offers a highly sensitive analytical platform to quantify trace constituents in complex biological matrices by selectively delivering analyte ions from an ESI source to an MS detector. Further sensitivity improvements with LC-MRM approach are achievable by enabling higher efficiency transport of analyte components from an ion source to the MS analyzer, eliminating dead times in analyses of fragment ions and reducing background ion signals. Electrodynamic ion funnel (IF)1or S-lense2 have been shown to drastically improve MS sensitivity. A recently introduced ion funnel trap (IFT) has been demonstrated to further enhance the limit of detection for both the time-of-flight3 and ion mobility time-of-flight mass spectrometers4.The premise for sensitivity improvement with the IFT coupled to a triple quadrupole instrument is due to: i) ion accumulation in the RF-energized trap, which facilitates improved droplet desolvation manifested in the reduced background ion noise at the detector, ii) enhancement in signal amplitude for a given transition because of an order-of-magnitude increase in the ion charge density per unit time compared to the continuous mode of operation, and iii) the unity duty cycle in signal detection, as the use of the trap eliminates dead times between transitions, which are inevitable with continuous ion streams.This work reports on implementation of LC-IFT-MRM analysis of trace constituents from a complex biological matrix using a commercial triple quadrupole instrument (TSQ, Thermo Fisher Scientific).
Chemicals and Materials: Lyophilized Kemptide, Angiotensin I, Syntide 2, Bradykinin, Leucine and Enkephalin, Dynorphin A Porcine 1-13, Neurotensin, and Fibrinopeptide A were purchased from Sigma-Aldrich (St. Louis, MO). These were serially diluted to prepare concentrations ranging from 0.25 nM to 500 nM in 0.25 mg/mL of tryptic digest of Shewanella oneidensis MR-1 proteins.
Figure 1. Experimental setup and graphical interface of instrument control software for an ion funnel trap
Figure 2. Pulsing sequence of experiment with the ion funnel trap. Ions are accumulated in the trap during dead time and dwell time followed by a 0.5 ms release event. Ion release event is synchronized with the start of Q3 scan.
Parent Ions
Ion Trans. Frag. Trans. Frag. Trans. Frag. CE
Kemptide386.74
(2+) 409.27 b3-NH3 539.34 a5-NH3 567.33 b5-NH3 25
Angiotensin I 432.90 (3+) 534.27 b4 619.36 a5 647.35 b5 21
Syntide 2503.32
(3+) 283.18 b3 429.28 y4 705.94 y142+ 23
Bradykinin 530.79
(2+) 522.27 y92+-NH3 710.36 y6 807.42 y7 30
Dynorphin A Porcine 1-13535.34
(3+) 455.21 y113+ - NH3 529.70 y13
3+ -NH3 712.68 y123+-NH3 28
Leucine Enkephalin556.28
(+) 278.11 b3 397.19 a4 425.18 b4 22
Neurotensin558.31
(3+) 578.85 y92+ 643.73 y10
2+ 725.90 y112+ 26
Fibrinopeptide A768.85
(2+) 445.25 y5 645.33 y7 1077.53 y11 29
Electronics to control IF and IFT
Source interface
TSQ
4 column HPLC
Timing pulse to synchronize trap release and Q3 scan
Voltages to control trap
Transition 1 Transition 2Deadtime
Deadtime
Deadtime
accumulation accumulation accumulation
eject eject ejectEntrance grid
Exit grid
Transition 3
Q3 Trigger
Exit grid Entrance grid
ION FUNNEL TRAP Q1 Q3Collision cellQ2
Detector
ION FUNNEL
10 torr 1 torr 1.5x10-3 torrmulti inlet
-1
-0.6
-0.2
0.2
0.6
1
0 5 10 15 20 25RETENTION TIME, min
INTE
NSI
TY
ION FUNNEL TRAP2 nM Anigiotensin I in 0.25 mg/mL Shewanella digest
ION FUNNEL 2 nM Anigiotensin I in 0.25 mg/mL Shewanella digest
0 5 10 15 20 25 30 350.000E+00
1.000E+08
2.000E+08
3.000E+08
4.000E+08
5.000E+08
6.000E+08
R² = 0.994070172854722Kemptide
fmoles loaded onto LC column
Peak
Are
a
0 5 10 15 20 25 30 350.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
R² = 0.997333156050591
Angiotensin I
fmoles loaded onto LC column
Peak
Are
a
0 5 10 15 20 25 30 350.00E+00
5.00E+08
1.00E+09
1.50E+09
2.00E+09
2.50E+09
R² = 0.996003925872965Leucine Enkephalin
fmoles loaded onto LC column
Peak
Are
a
Peak
Are
a
0 5 10 15 20 25 30 350.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
7.00E+08
R² = 0.994467325150108
fmoles loaded onto LC column
Fibrinopeptide A
5 10 15 20 25 30 350E+00
1E+08
2E+08
3E+08
4E+08
5E+08
6E+08
R² = 0.998842838158562
Neurotensin
fmoles loaded onto LC column
Peak
Are
a
0 5 10 15 20 25 30 350.00E+00
4.00E+07
8.00E+07
1.20E+08
1.60E+08
2.00E+08
R² = 0.99828682610235
fmoles loaded onto LC column
Bradykinin
Peak
Are
a
Figure 3. LC-MS/MS experiment with a 0.25 mg/mL Shewanalla oneidensis digest spiked with nine peptides (see table 1). Inset shows three transitions of neurotensin acquired in continuous (blue trace) and trapping (red trace) modes
SN
R
SN
R
kemptideLeucine Enkephalin
moles moles
Figure 4. Signal-to-Noise (SNR) of kemptide and leucine enkephalin as a function of the peptide amount added to a 0.25 mg/mL Shewanella oneidensis digest in LC-MRM experiments. Bottom panels show selected ion chromatograms representing three transitions of the two peptides and the matrix signal in the corresponding m/z ranges.
Table 1. List of peptides and the corresponding fragments monitored in LC-MRM experiments with a 0.25 mg/mL Shewanella oneidensis digest
0
20000
40000
60000
80000
534.265 534.266 534.267 534.268
m/z
INTE
NSI
TY
Angiotensin I, b4+, m/z 534.27ION FUNNEL TRAP
ION FUNNEL
0
20000
40000
60000
80000
619.356 619.357 619.358 619.359
m/z
INTE
NSI
TY
Angiotensin I, a5+, m/z 619.36
ION FUNNEL TRAP
ION FUNNEL
0
20000
40000
647.351 647.352 647.353 647.354
m/z
INTE
NSI
TY
Angiotensin I, b5+, m/z 647.36
ION FUNNEL TRAP
ION FUNNEL
Figure 5. Comparison of the selected ion chromatograms corresponding to the three transitions of Angiotensin I added with other 8 peptides to a 0.25 mg/mL digest of Shewanella oneidensis in LC-MRM experiments. Trapping time in the IFT was 40 ms.
Figure 6. Peak area as a function of the peptide amount for several peptides added to a 0.25 mg/mL Shewanella oneidensis digest in LC-IFT-MRM experiments.
